Accepted Manuscript / Manuscrito Aceptado

Tittle Paper/Título del artículo:.

Effect of chitosan on the in vitro control of Colletotrichum sp., and its

influence on post-harvest quality in Hass avocado fruits

Efecto del quitosano en el control in vitro de Colletotrichum sp., y su

influencia en la calidad poscosecha en frutos de aguacate Hass

Authors/Autores: Xoca-Orozco, L. A., Aguilera-Aguirre, S, López-García, U.

M., Gutiérrez-Martínez, P., Chacón-López, A.

ID: e355

DOI: http://dx.doi.org/10.15741/revbio.05.01.13

Received/Fecha de recepción: September 26th 2017.

Accepted /Fecha de aceptación: January 29th 2018.

Available online/Fecha de publicación: November 12th 2018.

Please cite this article as/Citar como: Xoca-Orozco, L. A., Aguilera-Aguirre, S, López-García, U. M.,
Gutiérrez-Martínez, P., Chacón-López, A. (2018) Effect of chitosan on the in vitro control of
Colletotrichum sp., and its influence on post-harvest quality in Hass avocado fruits. Revista Bio
Ciencias 5(1), e355. doi: 10.15741/revbio.05.01.13

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1
 Effect of chitosan on the in vitro control of Colletotrichum sp., and its
influence on post-harvest quality in Hass avocado fruits

Efecto del quitosano en el control in vitro de Colletotrichum sp., y su

influencia en la calidad poscosecha en frutos de aguacate Hass

Luis-Angel Xoca-Orozco1, Selene Aguilera-Aguirre1, Ulises Miguel López-García 1,

Porfirio Gutiérrez-Martínez*1 and Alejandra Chacón-López*1.
1 Instituto Tecnológico de Tepic. LIIA. Laboratorio de Biotecnología. Av.
Tecnológico #2295 Lagos del Country. Tepic, Nayarit, México.

Corresponding authors*: mchacon@ittepic.edu.mx, pgutierrez@ittepic.edu.mx

Resumen

El aguacate Hass, es desde el punto de vista económico, uno de los frutos más
importantes para México ya que es el principal productor en el mundo. Sin
embargo, la alta producción se ve afectada considerablemente ya que se
producen grandes pérdidas poscosecha debido a que el fruto es susceptible al
ataque por Colletotrichum sp., agente causal de la antracnosis. En los últimos
años se han explorado nuevas alternativas para combatir los hongos poscosecha
debido al riesgo que implica el uso de fungicidas químicos que son nocivos para el
medio ambiente y la salud del consumidor. Una de estas alternativas es la
aplicación de películas de quitosano, el cual posee un efecto antifúngico. En este
estudio se evaluó la actividad antifúngica in vitro de quitosano de bajo y medio
peso molecular en dos cepas de Colletotrichum sp., que fueron aisladas de frutos
de aguacate Hass con síntomas de antracnosis. El efecto del quitosano sobre la
calidad poscosecha en frutos de aguacate también fue evaluado. Se utilizaron
soluciones de quitosano al 0,1, 0,5, 1,0, 1,5 y 2,0% (p/v) para evaluar la inhibición
en el crecimiento micelial, esporulación y germinación de esporas, registrando las
mediciones cada 24 h durante 9 días. Los ensayos in vivo se llevaron a cabo
inoculando frutos de aguacate con una suspensión de Colletotrichum sp (1x106

2
esporas·mL-1). Los frutos fueron sumergidos en una solución de quitosano de bajo
peso molecular (1.5 % p/v). Se evaluó en los frutos tratados la incidencia de la
enfermedad, la pérdida fisiológica de peso, la firmeza y el porcentaje de materia
seca después de 12 días de almacenamiento a 25ºC. Los resultados in vitro
demostraron que tanto la solución de quitosano de bajo como de medio peso
molecular al 1% inhibieron más del 90% del crecimiento micelial y redujeron
significativamente la esporulación y la germinación de los conidios de
Colletotrichum sp. Los resultados in vivo mostraron que la aplicación de quitosano
en frutos de aguacate redujo la pérdida fisiológica de peso, mantuvo la firmeza y la
incidencia de la enfermedad fue disminuida. De acuerdo con nuestros resultados,
la aplicación de la película de quitosano en frutos de aguacate puede ser una
alternativa recomendable para controlar la antracnosis y preservar la calidad del
aguacate Hass durante su almacenamiento a temperatura ambiente.

Palabras clave: aguacate, quitosano, antifúngico, antracnosis, in vitro, in vivo.

Abstract

Avocado fruit cv Hass, is from an economic point of view, one of the most
important crops for Mexico, because Mexico is the main producer of avocado in the
world. However, the production is often reduced due to large postharvest losses
because the fruit is susceptible to attack by Colletotrichum sp., the causal agent of
anthracnose. In recent years it has been explored new alternatives to combat
postharvest fungi because of the risk that involve the use of harmful chemical
fungicides on the environment and consumer health. One alternative is the
application of chitosan films, which has an antifungal effect. In this study the in
vitro antifungal activity of low and medium molecular weight chitosan on two strains
of Colletotrichum sp., isolated from Hass avocado fruits whit anthracnose
symptoms was evaluated. Additionally, the chitosan effect on the post-harvest
quality of the avocado fruit was also evaluated. Chitosan solutions at 0.1, 0.5, 1.0,
1.5 and 2.0% (w/v) were used to assess inhibition of mycelial growth, sporulation
and germination of spores recording measurements every 24 h for 9 days. In vivo
trials were performed by inoculating avocado fruits with a suspension of
3
Colletotrichum sp (1x106 spores·mL-1). The fruits were immersed in low molecular
weight chitosan solution (1.5% w/v). The incidence of disease, physiological weight
loss, firmness and percent of dry matter of the treated fruits were evaluated after
12 days of storage at 25 ºC. The in vitro results demonstrated that low and medium
molecular weight chitosan solution at 1% inhibited more than 90% of mycelial
growth and significantly reduced sporulation and germination of conidia of
Colletotrichum sp. The in vivo results showed that the application of chitosan on
avocado fruits reduced physiological weight loss, kept the firmness and decreased
disease incidence. According to our results, the application of chitosan films on
avocado fruits could be a plausible alternative to control of anthracnose and
preserve the Hass avocado quality during its storage at room temperature.

Keywords: avocado, chitosan, antifungal, anthracnosis, in vitro, in vivo.

1. INTRODUCTION
Avocado is one of the main agricultural products in Mexico; its production
represents an important source of income for local, national and international
markets because Mexico ranks first in worldwide production of avocado (Senasica,
2017). Avocado is particularly susceptible to damage caused by Colletotrichum sp.,
a pathogen responsible for approximately 20 % of postharvest losses (Rodríguez-
López et al., 2009). Chemical fungicides reduce these losses but may have a
negative effect on the environment and cause consumer health problems if
residues remain in the fruit. Therefore, fungicides of biological origin are attractive
alternatives (Ochoa-Ascencio, 2009; Tamayo, 2007; Lárez, 2008).

Chitosan is a biological fungicide that does not pollute the environment and does
not presents risks to the health (Martínez-Camacho et al, 2010, Gutiérrez-Martínez
et al., 2012). Chitosan, poly [ß (1-4) 2-amino-2-deoxy-D-glucopyranose], is a
copolymer of D-glucosamine units of N-acetyl-D-glucosamine derived from the
chitin deacetylation in alkaline medium (Monarul et al. 2011). The fungicidal activity
of chitosan is partially explained by its cationic character. Positively charged free
amino groups in acid media interact with the negative residues from the

4
macromolecules of the exposed fungal wall, inducing changes in the permeability
of the plasma membrane and thus alteration of its functions (Benhamou, 1992).
Chitosan may also inhibit the synthesis of some essential enzymes for the fungus
(El-Ghaouth et al. 1992) and cause cytological disorders, such as blistering or lack
of cell cytoplasm, as reported for spores of A. alternata, Colletotrichum sp. and
Fusarium sp., subjected to 1-2% chitosan solutions (Sánchez-Domínguez et al.,
2007; López-Mora et al., 2013; Bautista-Baños et al., 2012).

Chitosan coatings form a semipermeable film on the fruit surface that slows the
respiration rate, reduces weight loss, maintains overall quality and increases the
shelf life of the fruit (Romanazzi, et al., 2013). In other fruits, such as mango,
chitosan has decreased the incidence of fungal diseases, increased firmness and
abated the physiological weight loss (López-Mora et al., 2013). Correa‐Pacheco et
al., (2017) applied chitosan nanoparticles in combination with thyme essential oil,
finding a reduction in the incidence of C. gloeosporioides on avocado Hass,
moreover, fruit firmness was better maintained in comparison with untreated fruit.
However, publications about chitosan use in avocado fruits are scarce. For this
reason, the objective of this research was to determine the in vitro antifungal effect
of low and medium molecular weight chitosan on Colletotrichum sp, which is the
causal agent of anthracnose and is considered one of the main postharvest fungi
that affects Hass avocado fruit (Persea Americana Mill), and on the other hand,
evaluate the fruit quality during storage at room temperature.

2. Materials and Methods

2.1 Isolation of phytopathogens

Hass avocado (Persea americana Mill) fruits in physiological maturity stage were
obtained from an orchard located in the Town of Tepic, Mexico. Fungus
development was encouraged by placing fruits in chambers at a relative humidity
of 90-95% at 25 °C for 5 days. Once symptoms developed, injured tissue was
disinfected by immersion in sodium hypochlorite solution (2%) for 2 min, rinsed
with sterile water for 2 min and placed on filter paper to eliminate moisture.
Moisture-free tissue sections were plated on potato dextrose agar (PDA) (DIFCO,
5
cat:DF0013) and were incubated at 25 °C for 24 to 72 h. The fungus genus was
determined considering the taxonomic keys described by Barnett and Hunter
(1998).

2.2 Pathogenesis test of isolates in avocado fruits

For the evaluation of the pathogenesis of the isolates and verify the Koch's
postulates, avocado fruits were inoculated with the isolated strains. Prior to
inoculation, strains were pre-incubated for 8 days. A spore suspension was
prepared according to Sellamuthu et al., (2013). Spores were counted using a
haemocytometer and were adjusted to 1x106 spores·mL-1. Using an insulin needle,
40 µL of the suspension was inoculated through the skin of healthy fruits previously
washed with water. Inoculated fruits were then stored at 25 °C until the onset of
symptoms appeared. A comparison of symptoms between the non-inoculated
(control) and the inoculated fruits was performed (Morales-García y et al., 2009).

2.3 Preparation of chitosan solutions and treatments

Chitosan was prepared as follows: low molecular weight chitosan (LMW) (Sigma
Aldrich cat: 448869; viscosity 35 cps in 1% chitosan solution; 96.1% deacetylation)
and medium molecular weight chitosan (MMW) (Sigma Aldrich cat: 448877;
viscosity 590 cps in 1% chitosan solution; 80% deacetylation) were used to
prepare solutions at 0.1, 0.5, 1.0, 1.5 and 2.0% (w/v) in 2% acetic acid. Solutions
were placed under constant stirring for 24 h, and then pH was adjusted to 5.6 with
1N NaOH prior to the addition of 0.1 mL of Tween 80.

2.4 In-vitro evaluations

2.4.1 Application of treatments

An 8 mm disc of mycelium was taken from the fungal periphery after 8 days of
incubation at 26°C. Discs were placed in the center of petri plates containing the
PDA-Chitosan media at the corresponding concentrations (0.1, 0.5, 1.0, 1.5 and
2.0%). A plate of PDA without chitosan was inoculated with the fungus and used as
the control. The inoculated petri plates were incubated at 26 °C, for 9 days.

6
2.4.2 Evaluations of the antifungal effect

Evaluation of the antifungal effect of chitosan on the isolates strain was determined
mycelial growth in petri plates (MG) and inhibition of mycelial growth (IMG),
sporulation and germination of spores, according to the methodology proposed by
Cordova-Albores et al., (2014).

2.5 In vivo evaluations

2.5.1 Inoculation of Colletotrichum sp., in avocado fruits

Avocado fruits in physiological maturity and free of pathogens or insect damage

were collected from a local orchard. The fruits were washed with potable water and
dried at room temperature. Using an insulin syringe, 40 μL of a spore suspension
(1x106 spores·mL-1) was inoculated by penetrating 3 mm into the fruit peel. After 30
min, fruits were treated with both low and medium weight chitosan solutions
(López-Mora et al, 2013).

2.5.2 Treatment of fruits with chitosan

Briefly, fruits were immersed for 1 min into the best treatment resulting from the in
vitro test (LMW chitosan solution at 1.5 %, w/v). Fruits were then allowed to dry for
60 min and were stored at 25 °C for 12 days. Treatments were labeled as FCh
(fruits treated with chitosan, 8 fruits), Fc (control fruit without chitosan, 8 fruits),
FChP (8 fruits treated with chitosan and inoculated with the fungus) and FP (8
fruits inoculated with the fungus and without chitosan).

2.5.3 Incidence and severity of anthracnose

To evaluate the effectiveness of chitosan to inhibit symptoms and decrease

anthracnose incidence, 32 fruits ripened to intermediate stage (firmness between
60 and 80 N) were inoculated with the pathogen (as indicated in section 2.2). Half
of the samples (16 fruits) were treated with LMW chitosan solution (1.5%, w/v), and
the other half was left untreated. All fruits were stored at room temperature for

7
ripening. In order to evaluate the development of symptoms, fruits were cut in the
area of inoculation and disease development was measured. Those fruits that had
size spots >1 cm2 were considered contaminated (Bill et al., 2014). The % of
incidence of disease (ID) was measured using the following equation:

% ID = fruit diseased / total fruits * 100

For the assessment of severity of the infection, the peels from the mature fruits
previously treated, were removed and pictures of the fruits without peel were taken,
then, the images were processed using the ImageJ software (Schneider et al.
2012) to determine the percentage of the total damaged area. This parameter was
expressed as percentage of affected area with respect to the total area of the
fruit.

2.5.4 Evaluation of the quality parameters in avocado fruit

Some quality parameters were evaluated in fruit treated and not treated with
chitosan. The parameters were the physiological weight loss (PWL), fruit firmness
and percent of dry matter. The PWL was measured every 24 h during 12 days of
storage. To determine the PWL, 10 fruits per treatment (FCh, Fc, FChP and FP)
were weighed every 24 hours using a digital weight scale (Sartorius model BL
3100, USA), and the results were expressed as suggested by El-Ghaouth et al.,
(1991) and were calculated as follows:

% PWL = (Initial weight - Final weight) / Initial weight x 100.

Determination of fruit firmness was performed on 3 samples using a texture
analyzer (Shimpo FGE-50, USA), equipped with a punch probe of 5 x 10 mm
diameter. The penetration test was applied to 3 points along the fruit husk (ends
and middle). The result was expressed as to the average of the obtained values
expressed in Newtons (N) (López-Mora et al., 2013). The determination of dry
matter was conducted per each treatment on 3 fruits previously peeled and cut into
small pieces (<0.5 cm2) and placed in a microwave oven (General Electric
JES832WK, 800 W) until a constant weight was achieved. The initial and final

8
weights of each sample were used for the calculations (Wang et al., 2012). The
evaluation of the firmness and dry matter was done on days 0, 3, 6, 7, 8, 9 and 10
of storage.

2.6 Statistical Analysis

For the in-vitro evaluation a 2x6 bifactorial design was used. The factors were:
chitosan molecular weight (LMW and MMW) and concentration (0.1, 0.5, 1.0, 1.5
and 2.0%). Each treatment was repeated three times. The in vivo evaluations were
conducted using a univariate completely randomized block design; all in vivo
assays were performed by duplicate. In both cases (in vitro and in vivo) the results
were analyzed statistically by analysis of variance (ANOVA). The comparisons of
means were made by Tukey test (P<0.01) using the statistical package SAS
System V9.

3. RESULTS

3.1 Isolation and identification of pathogens and pathogenicity test

The isolated pathogenic strains obtained from the damaged tissue were identified
as AG1, AG2 and AG3. Strains AG1 and AG3 exhibited similar characteristics:
random mycelial growth and small, white, cottony mycelium (Figure 1a). Strain
AG2 showed different characteristics than AG3 and AG1, it exhibited whiter cottony
growth compared to the grey colony color of AG1 and AG3 and a white-yellowish
coloration was observed on the base of the petri-dish (Figure 1b). According to the
identification performed with the taxonomic keys, AG1 and AG3 belong to the
Colletotrichum genus, whereas AG2 matches the genus Pestalotia (Figure 1c),
which is not considered an important pathogen of avocado fruit and for this reason
it was not included in the future evaluations. Fruits inoculated with strains AG1 and
AG3 presented the symptoms of anthracnose disease described above. The
macroscopic and microscopic characteristics of the strains corresponded to those
observed in the initial isolated strains (data not shown), complying with Koch's
postulates.

9
3.2 Antifungal effect of chitosan
In vitro evaluation of MG and % IMG of the strains AG1 and AG3 in the presence
of both LMW as MMW chitosan solutions at different concentrations, demonstrated
that the lowest MG occurred at the highest concentrations (1.5 and 2.0% w/v) of
LMW (Figure 2) and that >90% IMG was observed under these treatments
irrespective of the fungus strain (Figure 3).

Similar results were obtained with the MMW chitosan solutions (P<0.05) (data not
shown). The data displayed an inverse relationship between the chitosan
concentration and sporulation. LMW chitosan reduced the sporulation of strains
AG1 and AG3 by 29 and 51%, respectively, whereas MMW chitosan reduced
sporulation by 47 and 45%, (Table 1). Chitosan concentrations of 0.1, 0.5 and
1.0% (w/v) decreased spore germination (Figure 4); this parameter was completely
inhibited in both strains AG3 and AG1 when 1.5 % LMW and MMW chitosan
solutions were used (Table 1).

3.3 Effect of chitosan on the incidence and severity of anthracnose in

avocado fruits

LMW chitosan treatment resulted in a decrease in the incidence of anthracnose

reducing it by more than 80% compared to untreated fruits. The results of this
study showed that in fruits treated with chitosan the severity of infection decreased
by more than 95% (Figure 5).

3.4 Effect of chitosan on avocado fruit

During the 12 days of storage, PWL of untreated (Fc) and inoculated fruits (FP)
was of 19.6 and 20.9%, respectively. Chitosan treated fruits (FCh and QChP)
exhibited weight loss of 15.5 and 16.2%, respectively (Figure 6).

Chitosan treatment (FCh and FChP) maintained fruit firmness during 10 days of
evaluation at values of 30.8 and 29.5 N, respectively. Untreated samples (Fc and
FP) exhibited a decrease in firmness up to values of 19.8 and 11.8 N, respectively
(Figure 7).

10
Chitosan treated fruits exhibited lower dry matter content until 9 days of storage
compared to untreated samples. At the end of the evaluated period, the percent of
dry matter of fruits treated with chitosan (FCh and FChP) was of 30 to 33%, and of
untreated fruits (Fc and FP) was of 33 and 28%, respectively (Figure 8).

4. DISCUSSION

Beside of two strains of Colletotrichum, colonies identified as Pestalotia sp. were

also isolated from lesions similar to anthracnose. This fungus is found mainly in the
leaves of avocado trees and causes dead of branches and leaf spots (Morales-
Garcia, 2009). Some authors do not consider this fungus as an important
postharvest pathogen due to its low occurrence in avocado orchards (Tamayo,
2007). However, it is commonly confused with C. gloeosporioides because it
causes similar symptoms in fruits, although Pestalotia sp develops only in the peel,
does not damage the pulp and is less apparent in mature avocado (Morales-
Garcia, 2009). In contrast, Colletotrichum causes the anthracnose disease in
avocado fruits forming dark, sunken, circular or ellipsoidal lesions with large
numbers of spores that form compact salmon, orange or pink masses. In Mexico,
C. gloeosporioides is the main cause of anthracnose in avocado, but also C.
acutatum has been isolated from orchards in the state of Michoacan (Guillén-
Andrade et al., 2007). Among C. gloeosporioides fungus a morphological genetic
and pathogenic diversity has been identified using isozyme and fragments of
randomly amplified polymorphic DNA (Montero-Tavera et al., 2010). Genetic
relationships between different monoconidial strains are influenced by the
geographic origin and the symptoms they produce. The strains isolated in our
study named AG1 and AG3 had different coloration at the base of the petri dish but
share the same shape and size of spores. The color difference may be attributed to
this genetic variability observed between isolates.

Both LMW and MMW chitosan had a significant effect on the growth of the fungus,
and the maximum inhibition achieved was over 90% (at 1.5 and 2.0% chitosan)
(Figure 3b). Salvador et al., (1999) reported 100% inhibition of Colletotrichum sp
and Diplodia sp., isolated from Hass avocado with anthracnose symptoms, using
11
chitosan solutions of 0.1 to 0.5% w/v, but the study did not specify the chitosan
characteristics. Chitosan concentration and characteristics (molecular weight and
% of deacetylation) are responsible for the inhibition of fungus growth, mainly
through its polycationic nature that interacts electrostatically with negatively
charged phospholipids in the membrane, forming spaces through which the
chitosan can penetrate and reach the cytosol (Palma-Guerrero et al., 2010). The
interaction of the free amino groups, which are positively charged in acid media,
with the negative residues of exposed macromolecules in the wall of the fungi,
change the permeability of the plasmatic membrane, resulting in an alteration of its
principal functions, such as the output of wastes and intake of nutrients (Benjamou,
1992). In other studies it was demonstrated that chitosan influences the output of
potassium, the pH of the medium and the activity of the ATPase in the cell
membrane of the fungus R. stolonifer (García-Rincón et al., 2010). The spaces
formed in the membrane may also allow the free passage of calcium, causing an
imbalance gradient that destabilizes the cell (Bautista-Baños et al., 2005, Palma-
Guerrero et al., 2010). Chitosan altered the outer and nuclear membrane
permeability and may also inhibit RNA and protein synthesis when it entering the
cell and can be able to interact with the DNA, interfering the transcription process,
in addition to the polycationic nature (Möller et al., 2004; Rodríguez et al., 2005).
Furthermore, there is evidence that chitosan induces marked morphological
changes such as cell disruption and structural alterations (López-Mora et al., 2013;
El-Ghaouth et al., 1992). Evidence of the in vitro effectiveness of chitosan is
described by López-Mora et al., (2013), who used 1% LMW solution achieved
inhibition of approximately 80% for A. alternata isolated from mango; by the other
hand Liu et al., (2007), achieved 100% and 90% of inhibition of mycelial growth of
Botrytris cinerea and Penicillium expansum, respectively, using 1% (w/v) chitosan
solution. Our results were more effective compared to those described by Bautista-
Baños et al., (2005), who reported only 10% of IMG for C. gloeosporioides, isolated
from papaya using 1 and 2% (w/v) LMW chitosan. Some authors suggest that the
molecular weight of chitosan influences its action mechanism, so that, higher
molecular weight reduces permeability of the double nuclear membrane (Liu et al.,

12
2001; Aranaz et al., 2009). However, our results did not show significant
differences between the LMW and MMW treatments. The contrasting results may
be partially explained by the genetic and morphological variability between different
monoconidial strains of C. gloeosporioides (Ramos-García et al., 2010). Both
chitosan solutions (LMW and MMW) had a % IMG >90%. Previuos studies
reported similar % IMG with low and medium molecular weight chitosan (Kong et
al., 2010).

For its part, sporulation is important during fungal growth. The chitosan
concentration affects sporulation, and alters the cell differentiation processes and
the spores production. All the chitosan treatments reduced sporulation up to 50%
compared to the control. Strain AG3 was more susceptible to chitosan treatment,
whereas AG1 showed more resistance. However, neither strain (AG1 and AG3)
showed significant difference. Bautista-Baños et al., (2005) studied the behavior of
two C. gloeosporioides strains isolated from papaya (Veracruz and Guerrero,
Mexico) subjected to LMW, MMW and high molecular weight chitosan (HMW) and
found a reduction in sporulation greater than 50%, but there was no effect of
polymer weight on this parameter. We found that spore germination was reduced
by chitosan regardless of the type. Spore germination of 0% was achieved with
1.5% chitosan solutions (Table 2). According to Ocampo et al., (2009) the
development, growth, sporulation, germination, differentiation and virulence in a
variety of fungi is mediated by signal transduction, specifically by protein kinase A
which, which regulates, the asexual sporulation of the fungus Mucor circinelloides
(Rodríguez-López, 2009). This process may be affected in mutants that lack the
regulatory subunit of this protein. Chitosan treatments may affect signal
transduction of the protein kinase A pathway and affect spore development by
impairing the structure, physiological and reproductive functions or by differentially
activating signaling pathways.

At the same time, spore germination was decreased by the effect of chitosan and
according to our results, it occurs independently of the type used but is affected by
chitosan concentration. As mentioned previously chitosan affects the integrity of

13
the fungi cell membrane, which can cause losses of intracellular material
(Zakrzewska et al. 2005). This could be a signal to inhibit the germination of those
spores that were formed.

Since that in vitro tests we did not find significant differences between LMW and
MMW, for the in vivo experiments we used LMW only. LMW treatment reduced
disease incidence and severity in comparison to untreated fruits. This result may
be attributed to the effectiveness of the chitosan elicitor-like substance that may
activate the fruit defense system (Benigne-Ernest et al., 2008). In previous studies,
we analyzed the global gene expression of interaction of the Hass avocado-
Colletotrichum-chitosan system, the transcriptomic analysis showed a broad
expression for genes participating in fruit defense response (Xoca-Orozco et al.
2017; Gutiérrez-Martínez et al., 2016). In addition to this, we also observed a high
expression in some genes related to epicatechin and AFD synthesis, compounds
that are related to the defense system of the avocado fruit against the attack of
Colletotrichum (Wang et al., 2004a; 2004b; 2006).

Chitosan films also act as barriers for oxygen and carbon dioxide, which reduce the
incidence of the disease (Miranda et al. 2003) by ensuring water permeability,
slowing gas exchange limiting the development of the pathogen due to low oxygen
and ethylene production (Xaun-Zhu, 2008). This coating effect also reduced the
physiological weight loss of chitosan-treated fruits during storage (Figure 4a).
Salvador et al., (1999) evaluated different chitosan films and observed that fruits
lost 10% of their physiological weight after 6 days of storage, which was similar to
our results.

During fruit ripening and storage, firmness gradually decreased due to changes at
the cell wall level, hydrolysis of peptides, cellulase, pectin methylesterase and
polygalacturonase enzyme activity, which in turn degrade high molecular weight
polymers such as cellulose and hemicellulose (Silveira, 2007). In fruits treated with
LMW chitosan these processes are delayed by the presence of the film resulting in
less firmness loss and an extension of the fruit shelf life. Chitosan may contribute
to the preservation of pectin links and the distribution of cellulose in the cell wall
14
preventing degradation and maintaining fruit firmness (Elsabee and Abdou, 2013).
Bill et al., (2014) applied chitosan films to avocado fruits and reduced the incidence
and severity of anthracnose disease, increased resistance to the pathogen and
also maintained the firmness, the content of phenolic compounds and the activity
of enzymes related to the defense system of the fruit.

Fruit dry matter is an important parameter to determine avocado ripeness and it

depends on the period of fruit harvesting. However, as maturity progresses there is
a gradual increase in dry matter, and when the fruit ripens in the field it decreases
(Wang et al., 2012). Our results showed that dry the matter of fruits treated with
LMW did not increased during storage but significantly (p < 0.05) decreased when
fruit reached maturity. Ozdemir and Cavallazzi (2004) reported that this effect
could be due to inactivation of acetyl-CoA carboxylase, a key enzyme in the
production of long chain fatty acid from acetate-14C. Chitosan probably affected
the function of this enzyme in avocado tissue, which caused this behavior. Further
studies are needed to verify the effect of chitosan in inactivation of acetyl-CoA
carboxylase in avocado fruits.

5. Conclusions

The in vitro antifungal effectiveness of chitosan was optimal at concentrations of

1.5% w/v irrespective of the molecular weight. This treatment allowed a high
percent of IMG and reduced sporulation and germination of both strains of
Colletotrichum sp. Statistical analysis revealed no significant difference (p <0.05)
between LMW and MMW but some differences among the evaluated strains of
Colletotrichum sp were observed.

Chitosan treatment maintained the postharvest quality of the fruit, reduced the
incidence and severity of disease and physiological weight loss, and maintained
the firmness during storage. We propose that chitosan films could be a plausible
alternative to preserve the avocado quality and lessen the anthracnose incidence.
Additional quality parameters such as CO2 and ethylene production and enzyme

15
activity should be evaluated during storage to determine the effectiveness of
chitosan films during storage.

Acknowledgement

The authors thanks the CONACYT for the fellowship number 262345 awarded to
the participating master student and the PROMEP ITTEP-PTC-004 for the support
given to this project.

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FIGURES
L

Figure 1. Detail of isolates; AG1: Colletotrichum sp. AG2: Pestalotia sp. AG3:
Colletotrichum sp. (a) Top view of the growth plate. (b) Back view of the
growth plate. (c) Detail of the conidia of the isolates (100x).

22
Figure 2. Effect of LMW chitosan at different concentrations on mycelia
growth of AG1. Note: a-d Means with the same letter are not significantly
different (p <0.05)

23
Figure 3. Effect of LMW chitosan at different concentrations on % inhibition
of mycelial growth of AG1. Note: a-d Means with the same letter are not
significantly different (p <0.05)

24
Figure 4. Development of the germination of Colleotrichum spores treated
with different concentration of LMW chitosan, during 24 hours of
incubation at 27°C.

25
Figure 5. Effect of chitosan in the incidence and severity of anthracnose in
avocado fruits. Letters means are significantly different between treated and
untreated fruits (P <0.05)

26
Figure 6. Effect of LMW chitosan 1.5% in physiological weight loss during
storage at 25 °C. FCh: fruits with chitosan. Fc: fruits untreated (control).
FChP: fruits treated with chitosan and inoculated with pathogen. FP: fruits
inoculated with pathogen without chitosan.

27
Figure 7. Effect of LMW chitosan at 1.5% on firmness fruit. FCh: fruits with
chitosan. N: Newton. Fc: fruits untreated (control). FChP: fruits treated with
chitosan and inoculated with pathogen. FP: fruits inoculated with pathogen
without chitosan.

28
Figure 8. Effect of LMW chitosan 1.5% in dry matter of avocado fruits. FCh:
fruits with chitosan. Fc: fruits untreated (control). FChP: fruits treated with
chitosan and inoculated with pathogen. FP: fruits inoculated with pathogen
without chitosan.

29
Table 1. Final sporulation and germination of LMW and MMW chitosan
treatments for AG1 strains and AG3

Sporulation (x107 spore·mL-1) % Germination

Treatment LMW MMW LMW MMW
% w/v AG1 AG3 AG1 AG3 AG1 AG3 AG1 AG3
3.83 ± 3.54 ± 4.05 ± 3.65 ±
Untreated 100 100 100 100
1.54 0.01 1.29 0.41
3.25 ± 3.42 ± 3.93 ± 3.54 ±
0.1 60Aa* 100Aa 50Aa 50Aa
0.01Aa* 0.06Aa 0.10Aa 0.03Aa
3.14 ± 3.31 ± 3.22 ± 3.04 ±
0.5 50Aa 50Ba 50Aa 33Aa
0.2Aa 0.52Aa 1.16Ba 0.79Ba
2.96 ± 1.77 ± 3.14 ± 2.60 ±
1.0 25Ba 50Ba 50Aa 25Ba
0.25Ba 0.01Ba 0.02Ba 0.08Ca
2.73 ± 1.71 ± 2.13 ± 1.99 ±
1.5 0Ca 0Ca 0Ba 0Ca
0.05Ca 0.13Ba 0.16Ca 0.83Da
2.62 ± 1.47 ± 1.71 ± 1.80 ±
2.0 0Ca 0Ca 0Ba 0Ca
0.4Ca 0.13Ca 0.01Ca 0.48Da

**Reduction 29% 51% 47% 45% 100% 100% 100% 100%

Note: * Equal capital letters do not represent significant difference between the
different concentrations (rows). Equal lower letters do not represent significant
difference between the different strains (columns) (P <0.05). Mean ± SD. (n = 3).
** Reduction of sporulation and % germination compared to control and the
treatment of 1.5 % w/v of chitosan.

30